Chimeric poxvirus comprising sequences of a retroviral vector component

ABSTRACT

A chimeric poxvirus is disclosed, which comprises the sequences of the vector component of a retroviral vector particle and optionally the packaging component of a defective retroviral particle. The chimeric poxvirus according to the invention particularly can be used for applications in gene therapy or tumor therapy.

The invention relates to a chimeric poxvirus comprising the vectorcomponents of a retroviral (defective) genome. Furthermore, theinvention relates to a chimeric poxvirus comprising the vectorcomponents and packaging components for producing retroviral defectiveviruses, as well as the use of the chimeric poxvirus according to theinvention for gene or tumor therapy.

A central object of gene therapy is the stable genetic modification oftarget cells, and hitherto primarily viral vectors based onreplication-deficient adenoviruses and retroviruses have beenconstructed for application in gene therapy.

The in vivo gene transfer with adenoviral vectors is very efficient,since particularly with adenoviral vectors, a large number of differentcells can be transduced. However, in adenovirus infections, the DNA isnot integrated into the host genome, but is present in episomal form.Moreover, a cellular immune reaction can be triggered which restrictsthe foreign gene expression (Li et al., 1993, Hum. Gene Ther. 4:403-409). An attenuation of this reaction is possible by means ofvectors in which the adenovirus E3 region has not been deleted (Ilan etal., 1997. Proc. Natl. Acad. Sci. USA 94: 2587‥2592). Thesemodifications allow for a transient expression of the therapeutic genelasting for months. A permanent action, however, cannot be obtained withadenovirus vectors, since, as stated above, these vectors are notintegrated into the genome of the host cell in a stable manner.

In contrast thereto, retroviral vectors are capable of integratingforeign genes into the genome of the host cell in a stable manner.Retroviruses belong to the few presently known viruses which havemechanisms for integrating foreign DNA into the genome of the host celland thus are capable of permanently transforming the latter. On accountof this property, they are frequently used in gene therapy approaches.For this purpose, in techniques used so far, foreign genes have beencloned into the proviral DNA of retroviruses, wherein, however,replication-deficient retroviruses have been used, in which at least oneof the retroviral genes encoding for the packaging function (gag-pol orenv) has been inactivated or deleted. These constructs contain thepackaging signal psi and the flanking Long Terminal Repeat (LTR)sequences of the provirus. These transcription units are introduced intothe cells with the plasmid vectors by conventional transfectiontechniques and commonly transcribed in RNA in the nucleus under thecontrol of the LTR promoter. On account of the packaging signal psi,such an RNA is accepted as genomic viral RNA and packaged in retroviralparticles.

To form infectious retroviral particles, in the simplest case, the geneproducts gag-pol and env must be present. These genes can be insertedinto the cells also without the packaging signal via plasmidtransfection, or they may be provided via a helper virus. For preparingretroviral particles, also cell lines in which the viral genes for thepackaging components gag, pol and/or env are integrated in a stablemanner and which express the same, can be transduced. Such cell linesare termed “packaging” lines and are described e.g. in WO 97/35996.

Plasmid constructs have been described, so-called “plasmoviruses”, whichencode all the necessary components for the formation of non-replicatingretroviral particles (Noguiez-Hellin et al. 1996, Proc. Natl. Acad. Sci.USA 93: 4175-4180).

The use of replication-deficient retroviruses does, however, notcompletely prevent the risk that the retroviral DNA recombines with thehelper genes, whereby it may come to the production ofreplication-competent viruses which possibly may have a tumorigenicpotential.

Although the transformation of cells with retroviral vectors is highlyefficient in vitro, difficulties are encountered at present ingene-therapeutical applications. Thus, transfection techniques as arerequired in the production of retroviral vectors via plasmid constructs,can only be carried out in vitro. The direct administration of theplasmid constructs for production of the defective retroviral particlesin the body is extremely inefficient. Likewise, retroviral particles invitro can be produced only up to comparatively low titers. On account oftheir instability, concentration of the particles is possible also to alimited extent only. The low titers and the instability of retroviralparticles thus frequently prevent the efficient transformation in vivo.Methods, such as the direct injection of the particles into the targetorgan, or after tissue removal, ex vivo-transformation and implantationof transformed cells are being attempted at present. Also, the targetedtransduction of certain target organs, the so-called “targeting”, isvery difficult in case of retroviral vectors. It has been attempted tochange the tropism of the particles by heterologous enveloping proteins(pseudotyping).

The wide-spread retroviral vectors based on simple retroviruses, suchas, e.g., Moloney Murine Leukemia Virus (MLV), are able to transduceonly cells in the division phase. Cell growth in many fully grown organsis, however, very low. In tests for hepatic gene therapy, it has thusbeen attempted i.a. to obtain growing and thus transformable tissue by apartial removal of the liver. Likewise, retroviral vectors based oncomplex retroviral viruses, such as, e.g., HIV (lentiviral vectors) havebeen constructed, since the latter can also transform non-dividing cells(Reiser et al., 1996. Proc. Natl. Acad. Sci. USA 93: 15266-15271).

To correct the defective genes in case of gene therapy, an efficientinsertion of the corresponding genes as well as long-term expression ofthe genes are particularly important. New approaches for preparingvectors for the stable in vivo-transduction thus take advantage of theprinciple of using viruses as the carriers for retroviral defectiveparticles. In doing so, particularly the large insertion potential withDNA viruses, such as herpes virus and adenovirus, are utilized. Thefurthest-developed approach of chimeric virus systems is the preparationof defective retroviral particles by co-infection of two recombinantadenoviruses which encode the retroviral vector and the packagingfunction, respectively, the retroviral vector and the packaging proteinsbeing introduced via the adenovector system, whereby these cells becometransient retroviral production cells, and the retroviral vectorparticles formed therewith can infect the neighbouring cells (Feng etal., 1997. Nat. Biotechn. 15:866-870, Bilbao et al., 1997. FASEB J.11:624-634).

This system has, however, several decisive disadvantages: The acceptingcapacity of foreign genes of adenovirus vectors is restricted to a fewkilobases so that it is difficult to unite all the functions forproducing a defective retrovirus (packaging components and retroviralvector components) in one adenovirus. Such a construct is, however, theprerequisite for an efficient in vivo gene therapy. Moreover, bothadenovirus and retroviral genes and genomes are transcribed in thenucleus of the host cell and replicated, respectively. This topographicvicinity makes recombination to wild type retroviruses probable. Inprinciple, this system also does not allow for a deletion of importantretroviral transcription control regions, since the latter are necessaryfor the transcription in the nucleus. Thus, the adenovirus/retrovirussystem cannot be attenuated to the desired degree, in terms of safetytechnique. The same holds for the herpes simplex amplicon system; sofar, the latter has only been described for the expression of retroviralstructural proteins, wherein cells containing a lacZ provirus have beeninfected with an HSV amplicon vector containing the packagingcomponents, and thus retroviral lacZ particles have been obtained(Savard et al., 1997. J. Virol. 71:4111-4117).

A disadvantage both with adenoviral and with retroviral vectors is inparticular that introns for improving the foreign gene expression or forstabilising vector RNAs in the transduced cell cannot be applied sincethese introns have already been removed during the vector production bynucleus-specific splice mechanisms.

For a viral virus vector as a chimeric retrovirus carrier, also splicingand polyadenylating signals must be located at the correct site in theretroviral defective virus genome so as not to lead to a defectivesplicing or to a premature chain termination, respectively, during thetranscription of the retroviral genomes to be transduced. If, forinstance, the nuclear polyadenylating signal is put in front of thesecond, downstream retroviral LTR promoter, this will lead to a chaintermination of the transcripts in the nucleus and to a retroviraldefective genome no longer capable of transduction. In case of theatopic presence of these signals, the adeno- or herpes virusesreplicating in the nucleus would not form a transducing defectiveretrovirus and cannot be made safer in this manner. The insertion ofretroviral LTRs in herpes virus may, moreover, produce oncogenicsubspecies from non-oncogenic herpes viruses (Isfort et al., 1992, Proc.Natl. Acad. Sci., USA 89;991-995). This is particularly possible becausethe life cycles of the herpes viruses are performed in the nucleus.

Also alpha viruses have been used as vectors for producing retroviruses.Coinfects of several Semliki Forest vectors obtained via RNAssynthesized in vitro resulted in infectious retroviral vector particles(Li et al., 1993. Hum. Gene Ther. 4:403-409). The natural tropism ofcarrier viruses could be used for the gene transfer into the respectivepreferred cell types and tissues.

It has been the object of the present invention to provide a vectorsystem which does not have the above-mentioned disadvantages and allowsfor an efficient formation of retroviral particles.

According to the invention, this object has been achived by providing achimeric poxvirus which comprises the sequences of the vector componentof a retroviral particle.

By “vector component”, in the present context a defective retroviralvector genome is understood which contains all the sequences necessaryfor the expression of the retroviral genome, including the packagingsignal psi, as well as the sequences encoding a foreign protein. In thechimeric poxvirus of the invention, the sequence for the vectorcomponent in particular comprises a modified retroviral genome in whicha foreign gene, in particular one (or several) sequence(s) encoding aforeign protein, antisense DNA, one (or several) ribozyme(s), are underthe transcriptional control of a nucleus-active promoter, in particulara β-actin, CMV or SV40 early promoter.

The foreign protein may be any desired protein, a protein forsubstitution therapy or tumor therapy being, however, particularlypreferred. Proteins suitable for substitution therapy may be plasmaproteins, such as, e.g., factor II, factor V, factor VII, factor VIII,factor IX, factor X, factor XI, factor XIII, protein C, protein S, vonWillebrand factor or erythropoetin. Proteins suitable for tumor therapyare tumor suppressor proteins, such as p53 or p73, or “suicidal genes”,such as HSV TK, immunostimulators, such as B7.

The poxvirus-, in particular the vaccinia virus transcription apparatusrecognizes neither the LTR promoter of the retroviral provirus DNA aspromoter, nor does it recognize the proviral RNA processing signals.Thus, according to a special aspect of the present invention, thesequences of the vector component are put under the transcriptionalcontrol of a poxvirus-specific promoter.

There, particularly preferred promoters are poxvirus promoters whichcontrol the expression of the early genes. With the chimeric vacciniavirus of the invention (Retrovac vector), these are particularly theearly promoters which comprise both natural and synthetic promoters, asdescribed, e.g., by Davison et al. (1989, J. Mol. Bio. 210:749-769) andPfleiderer et al. (1995, Protein Expr. Purif. 6:559-569).

To prepare such constructs, e.g., the U3-region from the 5′ end of theproviral DNA can be deleted and replaced by a poxvirus-specificpromoter. Likewise, the U5-region from the 3′ end of the proviral DNAcan be deleted and a TTTTTNT signal can be added at the end of the “R”region. The repeated regions “R” at the ends of the transcripts areessential to the reverse transcription and thus to the functionality ofthe vector particles. Initiation upstream of the normal transcriptionstart leads to the synthesis of a transcript having a non-repeated 5′end, and thus, as expected, to reduced vector titers. With a view to acorrect 5′ end of the RNA, the initiation site of the poxvirus promotoreach inserted in the prototype construct should thus be optimized ineach case. Optimisation of such constructs is within the generalknowledge of the skilled artisan and can be carried out without greatexpenditures.

With the chimeric poxvirus according to the invention, defectiveretroviral particles can be prepared in a simple manner by infection ofsuitable packaging cell lines, such as, e.g., described in WO 97/35996,which express the packaging components.

According to a special embodiment of the present invention, the chimericpoxvirus contains the sequences which encode the vector components andthe packaging components.

The chimeric poxvirus according to the invention, comprising both thevector component and the packaging component, is capable of liberatingrecombinant defective retroviruses in situ, since it expresses in thetarget cell both the genes for the packaging components and also atranscription unit which encodes a retroviral defective genome. Besidesretroviral replication signals and packaging signals, the retroviraldefective genome preferably also comprises the foreign gene to betransduced and expressed, which is controlled either by the promoter ofthe retroviral long terminal repeat (LTR) itself, or by a secondpromoter.

Within the present invention, by “packaging component” any genesnecessary for forming a retroviral vector, such as gag-pol and env, of aretrovirus are understood. For the construction of the chimericpoxviruses of the invention (so-called RetroVac vectors), simpleretroviruses, such as MLV, just as well as complex retroviruses, e.g.human immunodeficiency virus (HIV) can be used. Likewise, for changingthe host spectrum, heterologous enveloping proteins (VSV-G, e.g.) can beexpressed for the transforming retroviral particles, as has alreadypreviously been described for retroviral vectors. Generally, most of thefurther developments on retroviral vectors can also be applied toRetroVac vectors. This also includes the expression of foreign genesunder the control of tissue-specific promoters and vectors for thesite-specific integration of foreign DNA.

According to the invention, the gag, pol and env genes preferably areunder the control of a poxvirus promoter. Preferred are promoters havinga large early portion, in agreement with the expression characteristicof the poxvirus. The sequences gag/pol and env encoding the packagingcomponents can be expressed in one transcription unit or on separatetranscription units under the control of a poxvirus/vacciniavirus-specific promoter, wherein the transcription unit may beintegrated in an essential or non-essential region of the poxvirusgenome.

With poxvirus transcripts, splicing does not take place. Retroviralgenes whose expression encompasses splicing, thus, according to afurther aspect of the present invention, are cloned as intron-freereading frames into the chimeric poxvirus of the invention. Depending onthe retroviral system selected, this aplies to the genes env, tat, refor other ones.

With the chimeric virus according to the invention, defective retroviralparticles can be produced in a simple manner both in vitro and in vivo.So far, no system has been described in which all the components for adefective retroviral particle have been encoded and combined on onesingle carrier virus. This is an essential pre-requisite for theefficient application of such vectors in vivo.

The development of packaging vectors and delivery/amplification systemsof transducing defective retroviruses on the basis of chimericpoxviruses so far has not been described. What has been described,however, is the use of poxvirus vectors for the expression of retroviralcomponents, in particular of gag-pol and env of HIV in poxvirus vectors(Moss, 1996, Proc. Natl. Acad. Sci., USA 93: 11341-11348, Paoletti,1996, Proc. Natl. Acad. Sci., USA 93: 11349-11353). The purpose of thesestudies was, however, fundamental virological research and vaccinedevelopment. Thus, e.g., it is known that the expression of the HIV-1gag-pol reading frame in VV (vaccinia virus) leads to the formation ofpseudoparticles (Karacostas et al., 1989, Proc. Natl. Acad. Sci., USA86:8964-8967). Likewise, the co-expression of gag and env leads to theformation of HIV-like particles which are particularly suitable forvaccine development (Haffar et al., 1990, J. Virol. 64: 2653-2659). Alsothe double-expression of gag-pol and env in poxvirus vectors as SIVcandidate vaccine has been described (Hirsch et al., 1996, J. Virol.70:3741-3752).

As the vectors for the chimeric poxviruses according to the invention,in particular chordopox viruses are used, which also include virusesfrom the group of the orthopox viruses and of the avipox viruses (Moss,1996, Poxviridae: the viruses and their replication. In: Fields et al.(ed.) Fields Virology. Third Edition (3^(rd). Ed.) Vol. 2.Lippincott-Raven, Philadelphia, 2637-2671). Preferably, however, suchpoxviruses are used which infect mammalian cells, yet do not propagatetherein (non-replicating vectors). As the vectors, thus preferablyvaccinia viruses are used, in particular attenuated vaccinia viruses(Paoletti, 1996, Proc. Natl. Acad. Sci., USA 93: 11349-11353), ModifiedVaccinia Ankara (MVA), or defective vaccinia viruses, such as describedin WO 95/30018 and in Holzer et al. (1997, J. Virol, 71:4997-5002). Thelast-mentioned defective virus vector can easily be propagated to titersof 10⁸ plaque forming units (PFU)/ml and concentrated to titers of 10¹¹PFU/ml. Moreover, e.g., D4-deleted defective viruses remain in the earlyphase of replication, which leads to a lasting synthesis of early RNAs.Only early vaccinia virus (VV)-RNAs have defined 5′ and 3′ ends, whichis a basic pre-requisite for the synthesis of functional retroviralgenoms. A vaccinia virus defective particle which expresses the entiregenetic information for a retroviral vector causes the formation ofnon-replicating retroviral particles in the infected cell, whichparticles in turn have a transforming potential. Since, as stated above,particularly vaccinia viruses can be concentrated to titers of 10¹¹PFU/ml, the titer, based on the transformation (expressed in CFU/ml) isthus higher than conventional retrovirus titers (>10⁶-10⁷). By theexpression of retroviral particles in situ, furthermore all thetransforming particles released over time are relevant for thetransduction and not, as in with the in vitro production ofretroviruses, the transducing particles per volume of cell culturesupernatant.

According to a special embodiment of the present invention, anon-replicating vaccinia virus, such as described in WO 95/30018, servesas the carrier of the genetic information for non-replicating defectiveretroviruses. Both the modified retroviral genome which contains theforeign gene and the packaging signal psi, and the retroviral genesgag-pol and env are encoded on the genome of a defective vaccinia virus.The sequences gag/pol and env encoding the packaging component can beexpressed in one transcription unit or in several transcription unitsunder the control of a poxvirus/vaccinia virus-specific promoter and canbe integrated in an essential or non-essential region of the poxvirusgenome. The sequences of the vector component can be inserted as anautonomous transcription unit, also under the control of a poxvirus,preferably an early poxvirus promoter, in an essential or non-essentialregion of the recombinant virus, insertion in an essential region beingpreferred.

The DNA sequence TTTTTNT leads to a termination of the earlypoxvirus/vaccinia transcription. To efficiently express the retroviralsequences, thus TTTTTNT signals possibly present in the retroviralsequences or in the foreign gene, respectively, should be modified bypoint mutation without changing the amino acid context of the retroviralproteins or destroying the controlling signals on the RNA genome of thedefective retroviruses, such as e.g. psi, and the integration region“att”.

According to a special embodiment of the present invention, thus thesequences encoding the packaging components and the vector components,respectively, do not comprise any poxvirus/vaccinia virus-specific stopsignals.

Just as for the expression of the vector component sequence, in thechimeric poxvirus-RetroVac system of the invention it is preferred toput the vector component sequence under the transcriptional control of apoxvirus-specific promoter.

In the preferred chimeric defective vaccinia virus constructs describedin the present invention, downstream of the 3′ region a signal wasinserted for the termination of early vaccinia virus transcripts, andthus the 3′ end of the mRNAs forming differ from that of previouslydescribed retroviral vector genomes. Only with the so-called earlyvaccinia mRNAs, most of them have a defined 3′ end. Late vaccinia mRNAswhich constitute the major portion of the viral transcripts do notterminate at concrete signals, have heterogenous lengths and thus arenot suitable as retroviral vector genomes. D4-defective VV do not enterthe late phase of replication. This could be demonstrated by ³⁵Slabelling experiments of proteins and in Northern Blot experiments by anunexpectedly long lasting early expression (Holzer et al., 1997, J.Virol. 71:4997-5002). Thus they proved to be a tool unique amongpoxviruses, for the synthesis of defined retroviral genomes.

One advantage of the chimeric poxviruses according to the invention isthat they can be propagated and concentrated to very high virus titers.Moreover, the vectors derived from poxvirus, in particular vacciniavirus, are extremely stable, efficiently infect organs, such as liver orspleen, and produce transforming, yet not replicating, retroviralparticles directly in the target organ (in vivo amplification of theretroviral particles).

Since poxviruses are tissue-specific (primary affine organs), atransformation is effected by the chimeric poxvirus of the invention, inparticular the chimeric vaccinia virus, in a tissue-specific manner. Forinstance, the infection with the chimeric vaccinia virus (RetroVachybrid vectors) preferably takes place in those tissues which correspondto the tropism of vaccinia virus, and which also express the receptorsfor the retroviral enveloping proteins used. Via suitable combinationsof the vaccinia virus strain used and pseudotyping, a more stringenttargeting of both the therapeutic gene and of the retroviral vectors canbe achieved.

The chimeric poxvirus vectors, in particular the RetroVac vectors basedon defective vaccinia virus, combine the ability of retroviruses tointegrate foreign DNA in target cells in a stable manner with thetechnical advantages of poxvirus/vaccinia vectors. A main characteristicof the system is that when a host cell is infected by the chimericpoxvirus, in particular a RetroVac vector based on a vaccinia virusvector, the proteins necessary for the formation of functionalretroviral particles are expressed and the mRNAs containing the foreigngenes are transcribed and packaged as genomic RNA in retroviralparticles. These particles are not capable of propagating(replication-deficient), yet they do have a transforming potential.While the cells primarily infected with the chimeric poxvirus, inparticular with the chimeric vaccinia virus, will die, secondaryretroviral infection of further cells by the retroviral particles formedwill lead to a permanent integration of the retroviral sequences, andthus of the sequences encoding the foreign gene, into the cell genome.

The liberation of defective retroviral particles by the chimericpoxviruses of the invention has been surprising insofar as normally thetranscription of retroviruses as well as the capping of the genomic RNAsoccur in the nucleus (Coffin, 1996, Retroviridae: the viruses and theirreplication. In: Fields et al. (ed.) Fields Virology. Third Edition(3^(rd) Ed.) Vol. 2, Lippincott-Raven, Philadelphia, 1767-1847). In thechimeric poxvirus system shown, transcription and capping occur in thecytoplasm. The vaccinia virus-specific cap structures thus are noobstacle for a retroviral packaging. Surprisingly it has been found thattranscripts generated by the cytoplasmatic poxvirus/vacciniatranscription system are compatible with the retroviral transcriptionand replication system. This was unexpected insofar as thepolyadenylating signals which are recognized in the nucleus are locatedwithin the U3 and R regions of the provirus.

As a rule, poxviruses are lytic viruses, whereby cells which have beeninfected by a chimeric poxvirus will die in most instances. On accountof the lesions thus caused in the tissue, division of neighbouring cellscan becaused which thus become even more susceptible to retroviraltransformation by the retroviral particles formed. In case of ectodermalcells, this proliferation effect is increased by growth factors of thepoxvirus itself.

In a special embodiment, thus the chimeric poxvirus according to theinvention comprises sequences encoding a growth factor or a mitogen. Bythis, the proliferation effect of the neighbouring cells of thepoxvirus-infected cells can be increased by the expression of chimericpoxvirus.

In a further special embodiment, it is possible to construct RetroVacvectors on the basis of lentiviruses, in which, however, accessorylentivirus genes must be expressed in the VV carrier, which give thesystem the properties desired.

In contrast to plasmid transfection, chimeric adenovirus/retrovirusvectors or chimeric herpes virus/retrovirus vectors, expression byvaccinia virus occurs in the cytoplasm of the host cell. While after aplasmid transfection or in case of an adenovirus/retrovirus infectionthe transcription of the foreign genes occurs in the nucleus by aid ofthe cellulary transcription apparatus, the gene expression of vacciniavirus exclusively occurs in the cytosol, by means of a viraltranscription apparatus different from the cellular one. Thisconstitutes a decisive safety advantage of the poxvirus vector of theinvention insofar as by this the generation of replicating retrovirusesbecomes very unlikely. This approach for the first time allows for thearrangement of nuclear transcription signals on the retroviral genomeexclusively according to the point of view of optimal foreign geneexpression and of the safety in the transduced target cell, since thetranscription of genomic retroviral RNA by vaccinia virus occursindependently of the nucleus of the target cell. Likewise, introns canbe used to enhance the expression of a therapeutic gene, since theformer do not influence vector RNA expression in the vaccinia virussystem.

A further particular advantage of the virus vector system of theinvention based on a DNA virus replicating in the cytoplasm is that,contrary to viruses propagating in the nucleus, retroviral transcriptionunits may contain transcription signals which normally are not allowedor not possible in connection with the nucleus, since splicing does notoccur in the life cycle of a cytoplasmatically transcribing virusvector. Thus, in the RetroVac system, e.g., the retroviral packagingsignal psi can be flanked by splicing signals, which, after transductionof the retroviral defective genomes and transcription of the same in thehost will lead to RNAs which have no packaging signals and thus havelost an essential feature of retroviral genomes (cf. FIG. 2C). Thispossibility substantially increases the safety of the system of theretroviral defective genomes produced in the poxvirus system. Thefunctionality of the packaging signal outside of the wild type contexthas already been shown.

A further aspect of the present invention is that in case of acytoplasmatic transcription of the retroviral defective RNAs,established transcription units having intron-exon structure can beintegrated undamaged (without splicing) into the genome of the host viaretroviral transduction (cf. FIG. 2A). When establishing permanentlyexpressing cell lines, as a rule gene cassettes are transferred,consisting of promoter, open reading frame (ORF) of the foreign gene,intron and polyadenylating signal, cloned into a bacterial plasmid. Foran optimal mRNA production, introns are required in transcription units,in particular this has also been observed in transgenic animals. Most ofthe commercially available expression vectors for higher cells containempirically determined promoter-intron combinations within theirexpression cassettes; e.g., the CMV promoter/enhancer SV40 introncombination in vector pCMVβ (Clontech Laboratories, Palo Alto) hasproved successful. Such optimized units having intron-exon structurecannot be transduced via the hitherto known retroviral or chimeric,respectively, gene transfer vectors.

The RetroVac system according to the invention solves this problem,since on account of the missing splicing apparatus, no introns areremoved in the vaccinia virus during the transcription. Aftertransduction of the retroviral defective genome produced in the RetroVacsystem, the complete transcription unit thus can be integrated in thetarget cell (cf. FIG. 2A). This is particularly important at thetransfer of cDNAs difficult to be expressed for the purpose of genetherapy, such as those of the coagulation factor VIII, and allows forthe transfer of optimized promoter/intron combinations.

According to a special aspect of the invention, thus by the systemaccording to the invention defective retroviral particles comprising anintron-containing genome are provided.

An RNA processing signal which may be transferred into the target cellat a defined site merely by cytosolic transcription systems is aninternal polyadenylating signal which causes a defined termination ofthe transcript in the nucleus (FIG. 2B). Termination of the transcriptbefore completion of a complete retroviral defective genome in thenucleus, such as by transduction of retroviral defective genomesproduced in the RetroVac system, allows for a particularly safe genetherapy, since the foreign gene once integrated cannot be transducedfurther, because its transcript terminates prematurely. Likewise, thelocalisation of the packaging signal between splice signals (FIG. 2C) ispossible only in the RetroVac system, which, in the target cell, leadsto the transcription of retroviral defective genomes which lack thepackaging signal. Thus, in combination with internal polyadenylatingsignals, particularly safe gene cassettes can be transferred.

A further aspect of the present invention relates to a compositioncomprising a chimeric poxvirus of the above-defined type and apharmaceutical carrier.

The RetroVac system according to the invention can be utilized in allapplications for which a gene therapy with retroviruses is meaningful.It is suitable for the in vivo gene therapy for the treatment of plasmaprotein defects, in particular hemophilias (factor IX and factor VIIIdeficiencies) and erythropoetin deficiency. Administration may beintravenously or intramuscularly.

With an appropriate formulation, the stability of the chimericpoxviruses according to the invention also allows for an oraladministration of the vectors as sprays, which enables inhalationtreatment of cystic fibrosis.

A further application of the RetroVac vectors is tumor therapy. It hasbeen shown, for instance, that the transfer of so-called suicide genesinto tumors or their metastases has proven to be promising in animalexperimental models (Caruso et al., 1993, Proc. Natl. Acad. Sci., USA90: 7024-7028, Culver et al., 1992, Science 256:1550-1552). In suchexperiments, the HSV-TK gene which converts non-toxic nucleoside analogs(such as Ganciclovir) into toxic ones, was transduced by intratumoralinjection of packaging cell lines which in situ liberate defectiveretroviral particles, and subsequently a chemotherapy was carried outwith Ganciclovir. With the RetroVac vector of the invention, thussuicide genes can safely and more efficiently be administered.

Likewise, apoptosis-induced tumor-suppressor genes, such as, e.g., thep53 gene which is altered in more than 50% of the tumors, can beinserted as foreign gene in the RetroVac vector of the invention. At thegene-therapeutical transfer of these tumor suppressor genes, the tumorcells would stop their growth on account of the endogenous control.

An ex vivo cell transduction for tumor therapy (e.g. leukemia) likewiseis possible; furthermore, the direct intratumoral injection for cancertherapy.

To increase the safety of the system, vectors can be used which carrythe so-called suicide genes, such as, e.g., the herpes simplex virus(HSV) with an inserted thymidine-kinase gene, which would allow for achemotherapy, if there were an activation of oncogens in vivo due to thetransduction procedure.

The system can also be used for producing permanent cell lines, sinceretroviral particles allow for a particularly efficient transduction andsince on account of the transfer of RNA processing signals (introns,polyadenylating sites) optimal gene cassettes can be transferred. Inthis manner, the highly efficient transfer of gene cassettes has beenpossible for the first time, which in connection with screeningtechniques allows for the rapid identification of highly expressing cellclones.

A special aspect of the present invention thus relates to the use of thechimeric poxvirus according to the invention for producing a medicament,which in particular can be used for gene therapy and tumor therapy.

Within the scope of the present invention, defective retroviralparticles were obtained via the above-mentioned chimeric poxviruses,which are particularly characterized in that they still comprise anintron-containing genome.

The invention will now be explained in more detail by way of thefollowing Examples and the drawing figures, to which, however, it is notrestricted.

FIGS. 1(A-B) shows the schematic representation of the construction ofchimeric vaccinia virus. FIG. 1A shows the possible insertion positionof the packaging and vector components, which optionally may beintegrated in different regions of the poxvirus genome and which areunder the transcriptional control of a poxvirus promoter. FIG. 1B showsthe infection diagram of a cell with the poxvirus of the invention, andthe production of defective retroviral particles which in turn infectcells and integrate into the host genome in a stable manner.

FIGS. 2(A-C) shows a comparison of a chimeric poxvirus replicating incytoplasm with the system of adeno or herpes viruses, respectively,which replicate in the nucleus, in particular with a view to thesplicing mechanism.

FIGS. 3(A-B) shows the construction diagram of plasmid pTKgpt-LSXN.

FIG. 4 shows the construction diagram of plasmid pD4-SX.

FIGS. 5(A-B) shows the construction diagram of plasmid pTK-MLVg.

FIG. 6 shows the construction diagram of plasmid pTKgpt-VP-FIX.

EXAMPLE 1

Construction of the vaccinia virus (VV)-defective virus vd-LXSN.

In the retroviral defective virus genome of the proviral vector plasmidpLXSN (Miller et al., 1989, Biotechniques. 7:980-990) the early vacciniavirus (VV) transcription stop signals were removed, and the thus deletedgenome was cloned behind a strong VV early promotor and then insertedinto the thymidine-kinase (tk) locus of the VV defective virus strainvD4-vA (Holzer et al., 1997, J. Virol. 71:4997-5002). In doing so,retroviral defective genomes were transcribed in the early VV infectioncycle in the resultant VV defective virus vd-LXSN.

Construction of the plasmid pTKgpt-LXSN

The retroviral defective genome in plasmid pLXSN (Clontech Laboratories,Inc., Palo Alto, Calf.) has the following order of genetic elements:LTR-psi-MCS/SV40-neo-gene-cassette-LTR (LTR, long terminal repeat; psi,packaging signal for retroviral RNA; MCS, multiple cloning site;SV40-neo, SV40 promoter neomycin resistence gene cassette). Into the MCSof this construct, suitable foreign genes can be inserted; the SV40-neogene cassette serves as the selection marker by aid of which theretroviral transduced cells can be selected.

The proviral retroviral DNA in pLXSN has three TTTTTNT signals whichlead to a partial termination of the early transcription. Individualfragments were cloned by PCR and modified by mutagenesis (FIG. 3). Forthis, the following 3 PCR fragments were amplified by means of differentoligonucleotides, with the plasmid pLXSN as template, and in doing so,point mutations were introduced which modify the TTTTTNT signalspresent. The preparation of the fragments LX1, LX2 and LX3 was effectedas described in the following:

LX1: A 1.6 kb fragment was generated with the oligonucleotide primersoRV-5 (5′-TACGTACGGC GCGCCAGTCT TCCGATAG-3′) and oRV-6 (5′-GAACCGGTCGCCCCTGCGCT GAC-3′), wherein an SnaBI cleavage side was introduced byoRV-5 and an AgeI cleavage site was introduced by oRV-6.

LX2: A 1 kb fragment was generated with the oligonucleotide primersoRV-7 (5′-AGACGTCCCA GGGACTTTGG GGGCCGTATT TGTGGC-3′) and oRV-8(5′-AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAATAAAAT TAG-3′) and the TTTTTNTmotive was modified by the two primers by means of point mutations;oRV-7 binds in the region of an AatII cleavage site, oRV-8 in the regionof an SfiI cleavage site.

LX3: The 3′ end of the planned vector construct was generated as the 1.2kb PCR fragment with the oligonucleotide primers oRV-9 (5′-CGACCGGTTCTATTTGTCAA GACCGACCT-3′) and oRV-10 (5′-GCGGCCGCAA CTGCAAGAGGGTTTATTGGA-3′). oRV-10 binds at the 3′ end of the “R” sequence andintroduces a NotI cleavage site.

To modify a TTTTTNT signal in the neo reading frame, a mutagenesis wascarried out in plasmid PLXSN. The mutagenesis was made by means of theQuickChange™ mutagenesis kit of Stratagene and the primers oRV-35(5′-AAATAGAACC GGTCGCCCCT GCGCTGAC-3′) and oRV-9. The resultant plasmidwas named pLXSNmut (FIG. 3b) and comprises a PinAI cleavage site at themutagenised site.

The LX1 PCR product was cloned into the vector pCRII (Invitrogen), theplasmid which contained the PCR product in the desired orientation wasselected and termed pCR-LX1(b). The LX2 PCR product was cleaved with theenzymes AatII and SfiI and inserted into AatII and SfiI cleavedpCR-LX1(b). The resultant plasmid was identical to pCR-LX1(b) except forthe modified TTTTTNT signals and was termed pCR-LX4. The ligation of a1.4 kb SfiI/SacI fragment from the plasmid pLXSNmut in SfiI/SacI cleavedpCR-LX4 yielded pCR-LX4+. This plasmid contains the entire transcribedregion of the vector, except for the outermost 3′ end.

The correct 3′ end was inserted as 0.8 kb fragment from pCR-LX3 into theVV recombination vector pTKgpt-selP (Pfleiderer et al., 1995, ProteinExpr. Purif. 6:559-69). The plasmid obtained was termed pTKgpt-LX3 (FIG.3a). The plasmid vector pTKgpt-LXSN was obtained by cleaving a 2.7 kgSacI/SnabI fragment out of pCR-LX4 and cloning into pTKgpt-LX3 cleavedwith SacI and StuI, and, under the control of a strong VV promoter, itcontained the entire transcribed region of the vector pLXSN from thebeginning of the “R” sequence (Coffin, J. M., 1996, Retroviridae: Theviruses and their replication, p. 1767-1847. In: Fields et al. (ed.)Fields, Virology, 3^(rd) ed. Vol. 2, Lippincott-Raven, Philadelphia) inthe 5′ LTR up to the end of the “R” sequence in the 3′ LTR, wherein thethree originally present TTTTTNT motives are modified (FIG. 3b).

Construction of the VV defective virus vd-LXSN

To construct the VV defective virus vd-LXSN, RK-D4R-44.20 cells wereinfected according to the standard protocol with the defective-VV vD4-vA(Holzer et al., 1997, J. Virol. 71:4997-5002) and transfected with theplasmid pTKgpt-LXSN. Virus isolates were plaque-purified in RK-D4R-44.20cells under gpt selection (Falkner et al., 1988, J. Virol.62:1849-1854). The thus obtained VV defective virus vd-LXSN expressesthe entire defective retroviral RNA genome of pLXSN. The correct genomicstructure of vd-LXSN was proven in Southern blot and by PCR.

EXAMPLE 2

Production of defective retrovirus particles by means of the VVdefective virus vd-LXSN in the packaging cell line PT67.

The defective virus vd-LXSN contains and transcribes a retrovirusdefective genome (cf. Example 1). The cell line PT67 (obtained fromClontech Laboratories, Inc., Palo Alto, Calf.) expresses the gag-pol andenv genes of murine leukemia virus (MLV) which are needed for packagingretroviral defective genomes (Miller et al., 1996, J. Virol.70:5564-71). To check the hypothesis that defective VV in the presenceof gag-pol- and env-proteins can form defective retrovirus particles,PT67 cells were infected with vd-LXSN. Cultivation of the PT67 cells waseffected according to standard conditions, as described in RetroXpressSystem User Manual. Clontech Laboratories, Inc., Palo Alto, Calif.,1997. The infection with vd-LXSN was effected at 0.05 or 0.5 PFU(plaqueforming units) per cell. At various points of time (3 h to 72 hpost infection), the retroviral vector particles were titrated in thesupernatants of NIH-3T3 cells (ATCC CRL-1658) in the presence of 500μg/ml G418 (Miller et al., 1996, J. Virol. 70:5564-71). The retroviraltiters reached a maximum 6 h post VV infection and in this range wereeach independent of the vd LXSN infection dose. In a typical experiment,with a primary infection of 0.5 PFU vd-LXSN per cell, after 3 h 10² CFU(colony forming units), and after 6 h 2×10² CFU were counted per ml ofculture supernatant. Control experiments with the plasmid pLXSN resultedin 10² CFU-10³ CFU per ml, 48 h post transfection. The retrovirus titerswhich were induced by vdLXSN infection, dropped to 20 CFU/ml at 12 hpost infection, to rise again to a maximum of 2×10² CFU/ml at 48 h to 72h pi. This second rise was not dependent on the initial infection doseand is attributed to a retrovirus-mediated transformation of VVuninfected PT67. No retroviral particles were obtained aftertransfection of the plasmid pTKgpt-LXSN in PT67, which proves that theexpression of this construct, other than, e.g., with adenoviruscarriers, is possible only in the viral context. This example shows thatdefective VV are suitable as carriers of transducing retroviraldefective genomes. The result is surprising insofar as normally thetranscription of retroviruses as well as the capping of the genomic RNAsoccurs in the nucleus (Coffin et al., supra) . In the RetroVac systemshown, transcription and capping occur by means of vaccinia-encodedenzymes in the cytoplasma. The VV speficic cap structures apparently areno obstacle to a retroviral packaging.

EXAMPLE 3

Construction of the VV defective virus vd-e10A1

In Examples 1 and 2 it has been shown that defective VV which transcriberetroviral genomes, form functional defective retroviruses in packagingcell lines. In the following, the 10A1 env gene was expressed in the D4locus of VV, a further step for combining all the components for theformation of defective RV particles on one defective VV. Cloning waseffected in intermediate steps, since one TTTTTNT motive of the envregion had to be deleted (FIG. 4).

Construction of the plasmid pD4-SX

Plasmid pCR-SX3mut: the MLV env gene (env SX: hybrid from the strains4070A and 10A1) was amplified as the 2 kb PCR product with the primersoRV-37 (5′-TCAGGGTCGAC ATGGAAGGTC CAGCGTTCTC-3′) and oRV-38(5′-GACTTTCATG ACTATGGCTC GTACTCTATA GGCTTC-3′) from the cell line PT67(Miller et al. 1996, J. Virol. 70:5564-5571) and cloned into the plasmidvector pCRII. A TTTTTNT signal in the env reading frame was modified bymutagenesis on this plasmid. Mutagenesis was effected by means of theQuickChange™ mutagenesis kit of Stratagene, and the primers oRV-41(5′-GAATGTTGTT TCTATGCAGA CCACACGGGA CTAGTGAGAG-3′) and oRV-42(5′CTCTCACTAG TCCCGTGTGG TCTGCATAGA AACAACATTC-3′). The resultantplasmid was termed pCR-SX3mut. Plasmid pTK-SXmut: From plasmidpCR-SX3mut, the env reading frame was cleaved out as 2 kb fragment withthe enzymes BspHI and HindII and ligated into the NcoI- and StuI-cleavedplasmid pTKL-selP, whereby plasmid pTKSX-mut was obtained.

Plasmid pTKL-selP: In plasmid pTKgpt-selP (Example 1), an 0.3 kbfragment was replaced via the cleavage sites HindIII and NdeI by asynthetic linker with the oligonucleotides oRV-22 (5′-AGCTCGCAATTGATGCATCA CA-3′) and oRV-23 (5′-TATGTGATGC ATCAATTGCG-3′) and theadditional cleavage sites NsiI and MunI were inserted.

Plasmid pD4-SX: An expression cassette comprised of the strong VVpromoter selP and the env reading frame was cleaved out of plasmidpTK-SXmut with the enzymes MunI and NotI, and ligated intoMunI/NotI-cleaved pD4-vA (21). The resultant vector pD4-SX (FIG. 4) thuscomprises the described expression cassette between flanking sequencesof the VV-D4 locus. Integration of this DNA in VV thus yields defective,non-replicating recombinants which express the env gene.

Construction of the VV defective virus vd-e10A1

The homologous recombination of pD4-SX with the VV wild type WesternReserve (WR) strain was effected according to standard techniques inRK-D4R-44.20 cells. Plaque purification was effected in RK-D4R-44.20cells twice with gpt selection and screening for lacZ-positive plaquesand twice without selection and screening for lacZ negative plaques(Holzer et al., 1997, J. Virol. 71:4997-5002). The resulting isolateswere termed vd-e10A1. A control of the env expression was effected inthe Western blot. An MoMLV-specific antiserum was obtained from QualityBiotech/Camden, N.J. (SerumID: 81S000044 “disrupted virus”).

EXAMPLE 4

Construction of the VV defective virus vd-eg

The MLV gag-pol reading frame was inserted into the hemagglutinin genelocus of the VV vector vd-e10A1 (Example 3). Thus, a defective VV formswhich expresses both gag-pol^(MLV) and env^(10-A1) and is capable offorming pseudoparticles. This MLV packaging vector still comprises afree thymidine-kinase gene locus into which retroviral defective genomescomprising foreign genes suitable for gene therapy can be inserted (asdescribed in Examples 5 and 6).

Cloning is effected in intermediate steps, since three TTTTTNT motivesof the gag-pol region had to be deleted (FIGS. 5a and 5 b).

The MLV gag-pol reading frame (5.2 kb) was amplified via PCR from thepackaging cell line PT67 (Clontech Laboratories, Inc., Palo Alto,Calif.). For this, four PCR fragments (G1-G4) were amplified whichoverlap at their ends. By the primers, the three TTTTTNT motives presentwere modified, and compatible cleavage sites were generated withoutinfluencing the amino acid sequence.

Amplification of the fragment G1: With the oligonucleotide primersoRV-11 (5′-CCATGGGCCA GACTGTTACC ACT-3′) and oRV-12 (5′-CTGGATCCTCAGAGAAAGAA GGGTT-3′), an 0.8 kb fragment was generated from MLV gag-pol,wherein by means of oRV-11 an NcoI cleavage site and by means of oRV-12a TTTTTNT signal was mutated and a BamHI cleavage site was introduced.

Amplification of the fragment G2: With the oligonucleotides oRV-13(5′-ATTAACCCTT CTTTTTCTGA GGATCCAGGT-3′) and oRV-14 (5′-CATCCTTGAATTCAAGCACA GTGTACCACT G-3′, a 1.7 kb fragment was generated fromMLV-gag-pol, wherein a BamHI cleavage site was introduced by theoligonucleotide oRV-13, as well as an additional SspI cleavage sitewhich it has at its 3′ end; by oRV-14, an EcoRI cleavage site isintroduced.

Amplification of the fragment G3: With the oligonucleotide primersoRV-15 (5′-TGAATTCAAG GATGCCTTCT TCTGCCTGA-3′) and oRV-16 (5′-AACTAGTAGATATTTATAGC CATAC-3′) a 2 kb fragment was generated from MLV-gag-pol,wherein oRV-15 mutates a TTTTTNT signal and introduces an EcoRI cleavagesite; by oRV-16, a SpeI cleavage site and an additional NotI cleavagesite which it has at its 3′ end, are introduced.

Amplification of the fragment G4: With the oligonucleotides oRV-17(5′-ATATCTACTA GTTTTCATAG ATACCT-3′) and oRV-18 (5′-GCGGCCGCTTAGGGGGCCTC GCGGGTTA-3′), an 0.8 kb fragment was generated from MLVgag-pol, wherein a TTTTTNT motive was modified by oRV-17 and a SpeIcleavage site and by oRV-18 a NotI cleavage site were introduced.

Cloning of the plasmids pTK-MLVg and pHA-MLVg

The G2 PCR fragment was cloned into the vector PCRII (Invitrogen, Inc)(pCR-G2), and from this again isolated via the enzymes SspI and NotI(FIG. 5a). Ligation in the BalI- and NotI-cleaved vector pTKgpt-selPyielded the plasmid pTK-G2 (FIG. 5b). The G1 PCR product was cleaved outof pCR-G2 by means of the enzymes NcoI and BamHI, isolated, and ligatedinto the NcoI- and BamHI-linearized plasmid pTK-G2, whereby the plasmidpTK-G12 was obtained (FIG. 5b).

The G3 PCR product was ligated into the vector pCRII so as to obtainpCR-G3. By digestion with the enzymes BamHI and StuI, Klenow treatmentand religation, a 1 kb sequence was deleted from pCR-G3, and into thethus-obtained plasmid pCR-G3d, the 0.8 kb-sized G4 PCR product wasinserted via the cleavage sites SpeI and NotI. The resultant plasmid wastermed pCR-G3d4. Insertion of a 1.1 kb fragment obtained by digestion ofpCR-G3 with the enzymes SacI and EcoRV into the SacI-and EcoRV-cleavedplasmid pCR-G3d4 yielded the plasmid pCR-G34, which comprises the 3′half of the MLV gag-pol reading frame. A 3 kb fragment was digested viathe EcoRI and NotI cleavage sites, and was recloned into the plasmidpTK-12 by religation of pCR-G34. In the resultant plasmid pTK-MLVg, theentire MLV gag-pol reading frame downstream of the strong early-late VVpromoter selP is contained (FIG. 5b).

The plasmid pTK-MLVg was cleaved with NdeI (partially) and NotI, and theselP-gag-pol cassette was isolated as 5.3 kb fragment. The fragment wasKlenow-treated and ligated into the SnaBI-linearized VV integrationplasmid vector pHA-vA. The resultant plasmid was termed pHA-MLVg.

Construction and functionality test of the packaging vector vd-eg.

The homologous recombination of the plasmid-DNA pHA-MLVg and thestarting virus vd-e10A1 (Example 3), as well as the plaque purificationwere effected according to the standard protocol in RK-D4R-44.20 cells(Holzer et al., 1997, J. Virol. 71:4997-5002.). The gag-pol expressionwas confirmed in the Western blot. The gag-pol- and env-expressingisolates were termed vd-eg.

Furthermore, the functionality of the expressed retroviral genes wastested on 3T3 cell clones which had been obtained by transformation withthe retroviral vector pLXSN. For this, 3T3 cells were transformed withdefective LXSN retrovirus, as described in RetroXpress System UserManual. Clontech Laboratories, Inc., Palo Alto, Calf. (1997). FiveG418-resistant clones were isolated. The supernatants of these cultureswere not infectious, and thus could not cause a transduction of native3T3 cells. These lines were infected with recombinant viruses vd-eg(moi=1). After three days, the supernatants were harvested and used forthe transformation of 3T3 cells. The transformation experiments wereeffected as described by Miller et al. (1996. J. Virol. 70: 5564-5571).The thus transformed 3T3 cells were G418-resistant, the supernatants ofselected clones were not transforming. The transformation observed thuswas not caused by a re-activation of retroviruses capable ofpropagation. This experiment proves that expression of the retroviralgenes by the packaging vector vd-eg enables the packaging oftransforming retroviral defective genomes.

EXAMPLE 5

Construction of the RetroVac vector vd-egLXSN

With vd-egLXSN as the starting vector, a VV vector was constructed whichexpresses all the components necessary for the formation of transducingdefective retroviral particles. The preparation of vd-egLXSN waseffected via homologous recombination with the plasmid-DNA pTKgpt-LXSN(Example 1) and the packaging virus vd-eg (Example 4) according to thestandard protocol in RK-D4R-44.20 cells (cf. Example 1). Isolates werepurified in several plaque purification rounds in RK-D4R-44.20 cellsunder gpt selection and termed vd-egLXSN. With sucrose gradient-purifiedvirus stocks of vd-egLXSN, 3T3 and RK13 cells were infected (moi=1).After 6 h, the supernatants were harvested and tested for transformationof 3T3 cells analogous to Example 1. Depending on the cell line, thesupernatants of 10⁶ cells contained between 10^(2 and) 10³ CFUretroviral vector. This Example proves that the infection of wild typecells with the RetroVac vector vd-egLXSN suffices to expresstransforming retroviral vector particles.

Example 6

Construction of a RetroVac vector vd-egC9 which expresses dRV particleswith factor IX cDNA insert.

In the following experiment, the human coagulation factor FIX is clonedinto a RetroVac vector, and retroviral defective particles are producedwhich are suitable for the gene therapy of hemophilia B. For thispurpose, a gene cassette comprised of the cytomegalovirus (CMV)promoter/enhancer (Boshart et al., 1985. Cell. 41:521-530) and thecoagulation factor FIX-cDNA and genetic elements (the “FIX genecassette”, as described in EP 0 711 835), which allow for RNAprocessing, and inserted into the plasmid pTKgpt-VP. The resultantplasmid pTKgpt-VP-FIX is recombined in the tk locus of the packagingvector vd-eg, resulting in the RetroVac vector vd-egC9.

Construction of the plasmid pTKgpt-VP-FIX

The factor IX-cDNA (Kurachi et al., 1982, proc. Natl. Acad. Sci., USA,79:6461-6464) comprises a TTTTTNT motive, which was modified viamutagenesis with the primers omut.FIX-1 and omut.FIX-2 (FIG. 6). Theresultant plasmid was termed pCMV-FIXmut.

pTKgpt-VP. Analogous to pTKgpt-LXSN, a plasmid vector was constructed onthe basis of PLNCX for VV expression. A TTTTTNT motive in the neomycinresistance gene of PLNCX was mutagenized for this purpose (primer oRV-35and oRV-9). The right half of the retroviral vector sequence wastransferred from this mutagenized plasmid pLNCXmut via the cleavagesites EcoRI and XbaI into the vector pTKgpt-LXSN. The resultant plasmidwas termed pTKgpt-VP and comprises the elements VV-thymidine-kinase (tk)gene flank/VV promoter selP/ RV-‘R’/RV-u5/psi/Neo/CMV promoter/MCS/RVU3/RV-‘R’/TTTTTNT/VV-tk flank.

For constructing plasmid pTKgpt-VP-FIX, a SnaBI/HindIII fragment whichcomprises a part of the CMV promoter and the entire FIX-cDNA was putfrom pCMV-FIXmut into SnaBI/HindIII (partial digestion)-cleavedpTKgpt-VP.

Construction and test of the RetroVac vector vd-egC9.

The plasmid pTKgpt-VP-FIX was recombined in the packaging vector vd-egaccording to standard methods and plaque-purified (cf. Example 5). Theresultant virus was termed vd-egC9. Sucrose gradient-purified stocks ofvd-egC9 were prepared to infect 3T3 and RK13 cells (moi=1). After 3days, the supernatants were harvested and tested for transformation of3T3 cells analogous to Example 4. Depending on the line, thesupernatants of 10⁶ cells contained between 10² and 10³ CFU retroviralvector. Infection of half-confluent RK13 (ATCC CCL-37) or SK-HEP-1 (ATCCHTB-52) cells with the supernatants which contained retroviral FIXdefective particles, expressed functional factor IX (measured asdescribed in EP 0 711 385).

EXAMPLE 7

Infusion of the RetroVac vector vd-egC9 in rabbits, and detection ofhuman factor IX in rabbit plasma.

In the following experiment it is shown in the animal test that theRetroVac vector vd-egC9 permits the expression of factor IX in vivo.

New Zealand white rabbits were administered 10⁸ and 10⁹ pfu vd-egC9intravenously, and for a period of one month, blood was drawn everysecond day and human factor IX was quantitated in ELISA. Pre-infusionsera were taken to determine, whether or not the ELISA is specific forthe human factor IX. Human factor IX could be found in rabbit plasma atconcentrations sufficient for a hemophilia B therapy. The presence ofanti-human plasma protein antibodies was determined after four weeks inWestern blots. In a control experiment, (infection with the defectivevirus vD4-vA), no human factor IX could be detected).

What is claimed is:
 1. A chimeric poxvirus comprising a poxvirus genomecomprising stably inserted therein a replication defective retroviralproviral genome under transcriptional control of a promoter, wherein aforeign gene sequence under transcriptional control of a promoter isinserted into said retroviral genome.
 2. The chimeric poxvirus as setforth in claim 1, further comprising a packaging component sequence. 3.A chimeric poxvirus as set forth in claim 1, wherein said poxvirus is achordopox virus.
 4. A chimeric poxvirus as set forth in claim 1, whereinsaid poxvirus is an orthopox virus.
 5. A chimeric poxvirus as set forthin claim 4, wherein said orthopox virus is a vaccinia virus.
 6. Achimeric poxvirus as set forth in claim 6, wherein said vaccinia virusis a defective vaccinia virus.
 7. The chimeric poxvirus as set forth inclaim 1, wherein said retroviral genome is under transcriptional controlof a poxvirus promoter.
 8. The chimeric poxvirus as set forth in claim2, wherein said packaging component sequence is under transcriptionalcontrol of a poxvirus promoter.
 9. The chimeric poxvirus as set forth inclaim 7, wherein said poxvirus promoter is an early poxvirus promoter.10. A chimeric poxvirus as set forth in claim 8, wherein said poxviruspromoter is an early poxvirus promoter.
 11. The chimeric poxvirus as setforth in claim 1, wherein said retroviral genome sequence comprisesintrons.
 12. The chimeric poxvirus as set forth in claim 1, wherein saidretroviral genome sequence comprises an internal polyadenylationsequence.
 13. A defective retroviral particle comprising anintron-containing genome.
 14. A method for producing a defectiveretroviral particle comprising a foreign gene sequence, wherein themethod comprises infecting a cell of a packaging cell line with achimeric poxvirus comprising a poxvirus genome having stably insertedtherein a replication defective retroviral genome under transcriptionalcontrol of a promoter, wherein a foreign gene sequence undertranscriptional control of a promoter is inserted into said retroviralgenome, and wherein the infected cell produces a defective retroviralparticle.
 15. A method for producing a defective retroviral particlecomprising a foreign gene sequence, wherein the method comprisesinfecting a cell of a cell line with a chimeric poxvirus comprising apoxvirus genome having stably inserted therein a replication defectiveretroviral genome under transcriptional control of a promoter, wherein aforeign gene sequence under transcriptional control of a promoter isinserted into said retroviral genome and retroviral packaging componentsunder transcriptional control of a promoter, and wherein the infectedcell produces a defective retroviral particle.